We have studied the regulatory elements in the mouse mammary tumor virus long terminal repeat (MMTV-LTR) by using a transient expression system. Plasmids were constructed that contained the LTR upstream from the prokaryotic gene chloramphenicol acetyltransferase (CAT). After transfection into mouse cells and growth with or without hormone, the CAT activity in cell extracts was measured as a parameter of gene expression. In fibroblasts (LTK-minus cells) as well as in transformed mammary tumor cells (34i) which express MMTV-RNA, the levels of CAT expression were low. The presence of enhancers on the transfected plasmids however, raised the level of CAT-expression considerably. Hormone inducibility could be observed using plasmids with or without enhancers. Deletion of sequences from the 5 feet end of the LTR results in a decrease in CAT activity in cells grown in the presence of hormone and an increase in the CAT activity in cells grown in the absence of hormone. A deletion retaining 179 bp upstream of the cap site is constitutively active. This may suggest that a negative regulatory element is involved in hormone regulation. After substituting the MMTV promoter region with one derived from Moloney sarcoma virus (MSV), we still observed induction of gene expression by steroid hormones. This suggests the presence of a discrete hormone responsive region which can function independently from the MMTV promoter.